After extracting the total RNA sample, the quality of the RNA is generally assessed based on the gel electrophoresis pattern. Due to the propensity of RNA to form secondary structures, formaldehyde denaturing gel is commonly used for RNA electrophoresis, which accurately reflects the quality of the RNA.

I. Reagents:
DEPC (product of Sigma), MOPs (product of Bocherigmer), Formamide (product of Sigma), Bromphenol Blue (product of Sigma), EDTA-Na2, sodium hydroxide, sodium acetate, formaldehyde, glycerol, analytical pure, domestically produced.

II. Reagent Preparation:
(1) Treatment of DEPC water: Measure 2L of deionized water using a graduated cylinder. In a fume hood, add 2ml of DEPC to 2 liters of deionized water, resulting in a final concentration of 0.1% DEPC. Quickly cover the container, mix well, and then place it on a shaker at medium speed for at least 4 hours, followed by high-pressure sterilization. During sterilization, loosen the bottle cap and sterilize at 15 pounds for 20 minutes.
(2) 10 × FA Buffer (formaldehyde agarose buffer: 200 mM MOPs, 50 mM NaAc, 10 mM EDTA): Prepare 500ml by adding 3.4g NaAC•3H2O to a 1000ml beaker, then add 400ml of DEPC-treated deionized water, and stir until dissolved. Add 20.9g MOPs and dissolve on a magnetic stirrer. Then add 1.86g EDTA disodium salt, and dissolve on a magnetic stirrer. Adjust the pH to 7.0 using 1M sterilized NaOH (approximately 40ml of NaOH), and then make up to 500ml with DEPC-treated deionized water. Store in a brown bottle at room temperature in a light-protected environment.
(3) 5 × Formaldehyde Denaturing Gel Loading Buffer (5×loading buffer): Prepare 10ml. Preprepare a water-saturated solution of Bromphenol Blue. In a 15ml sterilized centrifuge tube, sequentially add the following components:
4.0ml 10 × FA buffer
3.1ml Formamide
2.0ml 100% glycerol
720ul 37% formaldehyde
80ul 0.5M EDTA (pH 8.0)
16ul water-saturated Bromphenol Blue
100ul DEPC water
Mix well, aliquot, and store at -20°C, with common storage at 4°C.
(4) 1× Formaldehyde Denaturing Gel Electrophoresis Buffer (1×running buffer): Mix 20ml 10×FA gel buffer, 4.0ml 37% formaldehyde, and 176ml water. Use in the electrophoresis tank, and generally replace this electrophoresis buffer after 3 electrophoresis runs.
(5) 1.2% Formaldehyde Denaturing Gel: Weigh 0.4g agarose, add 3.34ml 10×FA gel buffer, add 30ml DEPC water, microwave until dissolved, and visually inspect for no suspended particles. Cool to approximately 50-60°C, then add 600ul formaldehyde and pour into a 7.5×5.0 cm gel mold. Insert a suitable comb, and the gel is ready for use after standing at room temperature for approximately 30 minutes.

Experimental Procedure:
Generally, take 0.3g of total RNA, add 1/5 volume of 5×loading buffer, heat at 65°C for 5 minutes, and rapidly cool on ice to eliminate the secondary structure of RNA. It is recommended to add 0.5-1.0ul of ethidium bromide (EtBr, concentration 1.0 mg/mL) to the RNA sample before loading, rather than adding EtBr to the gel, to reduce background after electrophoresis. Pre-electrophoresis the prepared 1.2% formaldehyde denaturing gel in 1× formaldehyde denaturing gel electrophoresis buffer for 15 minutes. Electrophorese the RNA sample at 5-10 V/cm for 30 minutes. Figure 1 shows the electrophoresis pattern of yeast total RNA using the above method.

Figure 1: Formaldehyde denaturing gel electrophoresis of yeast total RNA. 1. Yeast total RNA (0.3g); 2. Yeast total RNA (0.5g); 3. RNA Marker (0.2g). The quality assessment standard for RNA is the presence of clear 26S and 18S bands, absence of degradation, and the brightness of the 26S band is approximately 1-2 times that of the 18S band.